Sources of mismeasurement of RNA knockdown by DNAzymes and XNAzymes

RNA-cleaving oligonucleotide catalysts composed of DNA and/or nucleic acid analogues (DNAzymes, modified DNAzymes and XNAzymes) are promising agents for specific knockdown of disease-associated RNAs. However, we and others have identified discrepancies between their apparent activity in vitro versus when transfected into cells. Here, using examples of catalysts targeting the codon 12 region of KRAS RNA - an unmodified DNAzyme based on the classic "10-23" motif, a modified DNAzyme ("10-23_v46") or an XNAzyme ("FR6_1_KRas12B") - we examine confounding effects including unintended activity during standard RNA work-up steps, leading to mismeasurement of knockdown. We find that catalysts are not irreversibly denatured by typical cell lysis reagents, nor fully degraded by typical DNase treatments, exacerbated by nuclease resistant modification chemistries. In standard RT-qPCR workflows, DNAzymes and XNAzymes were found to be capable of cleaving their target RNAs during (1) DNase treatment and (2) reverse transcription (RT) reactions, in both instances with enhanced rates compared with under quasi-physiological conditions, producing cleavage-dependent false positives. Furthermore, catalysts were found to site-specifically inhibit cDNA synthesis (i.e. producing cleavage-independent false positives) and in the case of DNAzymes also had the capacity to act as primers during RT, leading to an enhancement of target site cDNA as judged by digital PCR, producing (cleavage-independent) false negatives. These effects could be broadly mitigated by purification to remove catalysts at the point of RNA extraction, under denaturing conditions. We recommend that studies of oligo catalysts in cells must include a 0 h timepoint after catalyst delivery or transfection to assess the collective impact of these mismeasurements on a case by case basis.